Tyrosine kinase inhibitors (TKIs) are the new generation of anti-cancer drugs with high potential against cancer cells’ proliferation and growth. However, TKIs are associated with severe cardiotoxicity, limiting their clinical value. One TKI that has been developed recently but not explored much is Ponatinib. The use of nanoparticles (NPs) as a better therapeutic agent to deliver anti-cancer drugs and reduce their cardiotoxicity has been recently considered. In this study, with the aim to reduce Ponatinib cardiotoxicity, Poly-b-poly(ethyleneoxide)-b-poly (PLGA-PEG-PLGA) triblock copolymer was used to synthesize Ponatinib in loaded PLGA-PEG-PLGA NPs for chronic myeloid leukemia (CML) treatment. In addition to physicochemical NPs characterization  the efficacy and safety of these drug-delivery systems were assessed in vivo using zebrafish. Zebrafish are a powerful animal model for investigating the cardiotoxicity associated with anti-cancer drugs such as TKIs, to determine the optimum concentration of smart NPs with the least side effects, and to generate a xenograft model of several cancer types. Therefore, the cardiotoxicity of unloaded and drug-loaded PLGA-PEG-PLGA NPs was studied using the zebrafish model by measuring the survival rate and cardiac function parameters, and therapeutic concentration for in vivo efficacy studies was optimized in an in vivo setting. Further, the efficacy of drug-loaded PLGA-PEG-PLGA NPs was tested on the zebrafish cancer xenograft model, in which human myelogenous leukemia cell line K562 was transplanted into zebrafish embryos. Our results demonstrated that the Ponatinib-loaded PLGA-PEG-PLGA NPs at a concentration of 0.001 mg/mL are non-toxic/non-cardio-toxic in the studied zebrafish xenograft model. BCR-ABL oncogene, which would cause abnormal cell proliferation and complications in the patients [Cancer is the second leading cause of death worldwide with a high number of incidents . Cancer patients .Therefore, the demand for successful anti-cancer therapeutics and the development of effective tools for early cancer detection and screening have increased. For example, the introduction of TKIs  such as Due to the biocompatibility and biodegradability of the PLGA and PEG triblock copolymers, they have been extensively used in drug delivery ,13. MoreZebrafish have been used as a research model in many applications, such as cancer and neuronal disorders studies, due to their numerous unique characteristics ,20,21. FIn this study, we first developed and characterized PLGA-PEG-PLGA NPs and then loaded the generated PLGA-PEG-PLGA NPs with different concentrations of Ponatinib. Next, we tested these NPs efficacy in reducing the cardiotoxic effect of Ponatinib in the zebrafish xenograft model. According to our results, PLGA-PEG-PLGA NPs demonstrated their efficacy in reducing the well-known cardiotoxic side effects of Ponatinib in our studied model.4). After that to determine how much media were required to add into each T-75 flask for passaging the cells the following was followed:Human CML K-562 cell line was obtained from the American-type culture collection (ATCC)  and as a kind gift from Shahab Uddin Khan from the interim Translational Research Institute (iTRI) at Hamad Medical Corporation (HMC), Doha, Qatar. Cells have been cultured according to the optimum conditions described by the manufacturer. The cells were cultured in RPMI 1640 supplemented with 10% FBS, 10,000 U/mL Penicillin-Streptomycin, and 100× GlutaMAX at 37 °C in a humidified 5% CO2 incubator. All reagents were obtained from Gibco . The cells’ culture medium was changed every other day to obtain the optimum cell count and maintain their viability at 90% following this equation: No. of viable cells/total No. of cells × 100. The cell counting was performed by obtaining all the cell suspension from the T75 flasks into sterile (15 mL or 50 mL) tubes, then centrifuged at 1300 rpm for 5 min using centrifuge 5804 , the supernatant was discarded and then the pellet was re-suspended in 3–2 mL RPMI 1640 media. Then the cell count was performed manually using a KOVA™ Glasstic™ Slide 10 with Grids  by taking 20 μL of the cell suspension mixed with 20 μL of the trypan blue stain  and then 20 μL of the mixture was loaded in the hemocytometer. Only the cells in the large 4 squares at the edges were counted under a light microscope. After that, the cell count in an ml was done following the equation: cell count × dilution factor (2) × the hemocytometer constant (106 cells/mL), they have been harvested by pelleting using a centrifuge at 1200 rpm for 5 min, the supernatant was then discarded then re-suspended in 3 mL PBS mixed with 6 μL of 5 μg/mL CM-Dil fluorescent dye Invitrogen . Then, the labeled cells were incubated for 5 min at 37 °C followed by a 15–20 min incubation at 4 °C. After that, the cells were checked under the fluorescence microscope Olympus IX73  using fluorescent filters with excitation/emission spectra of 553/570 nm maxima.Once the K-562 cells have reached confluency (1 × 10Wild-type zebrafish embryos (AB strain) were used for this experiment. All animal experiments were carried out according to national and international guidelines for the use of zebrafish in experimental settings  and follZebrafish embryos were counted and investigated for survival and morphological changes in 24 h intervals for 3 days using Zeiss SteREO Discovery V8 microscope with a Hamamatsu Orca Flash high-speed camera and images were analyzed using HCImage software. Dead embryos were scored according to the opaque color which they exhibit. Observed abnormalities have been investigated and recorded. Non-viable embryos were eliminated as soon as they were observed, whereas embryos with abnormal development were kept till the endpoint of the experiment.After the incubation period, 72 hpf, six embryos from each experimental group were stabilized using 3% methylcellulose and visualized under the microscope. A 10 s bright field video of the beating heart and the body was recorded for each embryo at 100 frames per second (fps). The same region in the dorsal aorta (DA) and the posterior cardinal vein (PCV) was localized to measure the flow velocity, arterial pulse, and vessel diameter using Viewpoints MicroZebralab version 3.6 application ,21. The zebrafish embryos were exposed to Pronase for 10 min at 24 h post-fertilization to remove the chorion. After that, they were incubated till 2 or 3 days-post-fertilization (dpf) at 28 °C. Dechorionated embryos were transferred to an injection slide, and they were anesthetized with 1% Tricane solution  for destabilization. The fluorescently labeled K562 cells were then injected into the yolk sac to allow the cells to enter the blood circulation using a fashioned glass capillary needle . About 300 K562 cells were injected into each embryo, using the Harvard Apparatus PLI 90A picolitre injector  at the BRC zebrafish facility. The embryos were first anesthetized with 200 mg/L Tricane for 5 min and were aligned properly to have their body on one side to allow easier access to their yolk sacs. Then, a capillary needle was prepared using borosilicate glass microcapillaries following the setting on the Narishige PC-100 puller. A total of 10 µL of the cells’ solution was then loaded into the needle and the needle was placed into a manipulator. The manipulator was then adjusted until holding the needle at a 45° angle to an embryo. The needle tip was broken slightly with tweezers, and the cells’ solution was gently injected into the zebrafish embryos’ yolk sacs. After that, the xenotransplanted embryos were transferred into new plates and fresh egg water and kept at 34 °C till the endpoint at 7 dpf. The zebrafish larvae were imaged under the fluorescence microscope using the ZEISS ZEN Microscope software  each day after injection to check the cancer cell spread and to measure the tumor size.®® 20 Ultrafiltration Unit)  to wash and concentrate the NPs (three washing cycles with Milli Q water at 4500 RPM for 10 min).The Poly-b-poly(ethylene oxide)-b-poly PLGA- PEG- PLGA polymer used to generate the NPs was purchased from Akina Inc., USA. A total of 25 mg of PLGA- PEG- PLGA polymer , along with 5 mg of the fluorescently labeled  were dissolved in 10 mL of Tetrahydrofuran (THF) . To induce nanoprecipitation and embryonic NPs formation, the organic solution was transferred drop-by-drop into 20 mL Milli Q water containing 5 mg Pluronic F127 . The dispersion was kept overnight with a magnetic stirrer to evaporate the organic solvent. The next day, the NPs dispersion was filtered through a 0.45 micron filter, and the filtrate was placed in the ultrafiltration tube  has been used to characterize the PLGA-PEG-PLGA NPs. The procedure was carried out by the Central Laboratories Unit (CLU) at Qatar University, by depositing a large droplet (around 10 μL) from each NPs sample onto a TF20 holder, and images were then obtained using a voltage of 200 kV.The particles’ surface morphology was assessed using NOVA NANOSEM 450 (N-SEM)  by the Central Laboratories Unit (CLU) at Qatar University. SEM uses a field emission gun as a source of electrons. The electron beam then travels through the column while being adjusted by different lenses till reaching the sample. The electrons interact with the sample producing secondary electrons and characteristic X-rays that can be detected by a special detector to produce electron images and elemental spectra correspondingly.The size of PLGA-PEG-PLGA NPs has been measured by Zetasizer Nano ZS . The cuvette was filled with the NPs dispersion and inserted into the machine after selecting the corresponding refractive index of the NP.The surface charges of the loaded and unloaded PLGA-PEG-PLGA NPs were determined by the Zetasizer Nano ZS . The machine measures the Zeta potential by using electrophoretic light scattering. The PLGA-PEG-PLGA refractive index was obtained from the literature  and the To determine the dissolution rate of the loaded drug in the NPs, a dialysis membrane method was performed. This was performed using the Float-A-Lyzer G2 membrane (MWCO 20 kDa), which traps the particles inside and allows the loaded drug to be released into the surrounding media. The NPs solution of 1 or 0.5 mL has been loaded inside the dialysis tube which was placed inside a beaker filled with PBS buffer (pH 7.4) with a magnetic stirrer at 37 °C for 24 h. After that, samples were taken for HPLC analysis from the same spot of the PBS buffer at regular intervals  results are shown in .For the efficacy of and quantitative assessment of loading and drug content of encapsulated Ponatinib, the HPLC method was used. Adequate volume of Ponatinib loaded PLGA-PEG-PLGA NP dispersion was diluted with a mobile phase and the quantification procedure was performed as described below. The efficacy of encapsulation was determined using the following equation:Degree content was calculated using the equation:2PO4 0.0037 mol/L (40%), pH 3.5 adjusted by H3PO4; and: (B) Acetonitrile (60%). The quantification was performed using PDA/UV detector at 250 nm wavelength.Quantitative analysis was performed on WATERS ACQUITY UPLC system HPLC system, using a C18 column, the flow rate of 1.2 mL/min, and injection volume of 5 μL. The mobile phase was composed of: (A) KHThe zebrafish embryos at 24 h post-fertilization (hpf) were exposed to 200 µL Pronase solution (1 mg/mL)  for 10 min to remove the chorion. Dechorioned embryos were then evaluated under the stereomicroscope  and segregated into 6-wells plates equally (about 20 or 24 embryos in each well). After that, different concentrations of the unloaded NPs were prepared to determine the optimum concentration that will not cause any toxicity to the zebrafish embryos. Different concentrations of NPs have been prepared by diluting the proper amount of the NPs in (0.3 mg/mL) 1-phenyl-2-thiourea (PTU) in the egg water, the concentrations were: 1, 0.75, 0.5, 0.25, 0.1 mg/mL. The embryos then were incubated with different concentrations of NPs at 30 °C and the survival rate was then measured at 48 and 72 hpf.The zebrafish embryos at 24 h post-fertilization (hpf) were exposed to 200 µL Pronase (1 mg/mL) solution for 10 min to remove the chorion. Dechorionated embryos were then evaluated under the stereomicroscope and segregated into 6-well plates equally (about 20 or 24 embryos in each well). After that, three different concentrations of loaded NPs with Ponatinib  were prepared to select the least toxic concentration for the zebrafish embryos. Different concentrations of NPs in PTU containing egg water were prepared as follows: 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.01, 0.005, 0.0025 mg/mL to choose for loading the Ponatinib drug. The negative control group was untreated zebrafish embryos kept in egg water. The embryos were then incubated at 30 °C and the survival rate was measured at 48 and 72 hpf. The survival rate was calculated by dividing the number of viable embryos by the total number of embryos multiplied by 100.Injected 2-dpf zebrafish embryos were allowed to recover for half an hour after injecting of K562 cells before exposing them to 0.001 mg/mL loaded PLGA-PEG-PLGA NPs with 10 mg and 15 mg Ponatinib. The embryos were separated and placed into 6-well plates: two wells for each group  with 10 embryos in each. The 0.001 mg/mL concentration of loaded PLGA-PEG-PLGA NPs was prepared by diluting it in egg water. For a total volume of 3 mL, the required amount for one well, i.e., 1.440 µL of the 15 and 10 mg NPs were diluted in egg water. Then the embryos were incubated at 34 °C and started to be imaged at 4-dpf.On day 2 pf, the dead embryos were removed from the 6-well cell culture plates to avoid influencing the surviving embryos during the toxicity experiments. The numbers of the dead, surviving, and abnormal embryos of each NPs concentration group were recorded until 3 dpf. The survival rate was calculated by dividing the number of viable embryos by the total number of embryos, multiplied by 100.To assess the cardiovascular toxicity side effects of both unloaded and loaded NPs, the analysis was carried out at 3-dpf for the embryos in all the treated groups to see the influence of interference on cardiac function, structure, and blood flow. The treated embryos were first placed in a concave slide for imaging using 3% methylcellulose for immobilization. Under the Hamamatsu Orca high-speed camera and Zeiss Lumar V12 stereomicroscope , images and high-speed time-lapse movies were recorded at about 100 fps for the heart and tail of each embryo through the HCImage software . Then to assess for heart failure due to the toxicity of the NPs, tail videos have been analyzed for the Red Blood Cells (RBCs) movement within the blood flow using the MicroZebraLab . Tracking the RBCs aids in measuring the blood velocity by following an in-house algorithm from Viewpoint for tracking RBCs. This algorithm has also been used to measure heart rate in beats per minute. Heartbeat and blood flow velocity parameters are widely used to assess cardiac function in zebrafish. Lower heartbeat and/or blood flow velocity indicates deteriorated heart function.p-value of less than 0.05 was considered statistically significant. One asterisk (*) indicates p < 0.05, two asterisks (**) indicate p < 0.01, three asterisks (***) p < 0.001, and four asterisks (****) indicate p < 0.0001.Statistical analysis was performed using GraphPad Prism version 9.0.0 software . Data were analyzed using one way-ANOVA with Dunnet’s multiple comparison test. A Olympus fluorescent microscope was used to image the fluorescent K562 CML cells stained with CM-Dil fluorescent dye. The mCherry fluorescent filter with excitation/emission spectra of 587/610 has been chosen to examine the fluorescent K562 CML cells as the CM-Dil fluorescent dye has an excitation/emission of 553/570 nm maxima. In the course of the nanoprecipitation process, particles are generated by simultaneous polymer/drug nucleation, molecular growth, and aggregation during the micromixing of water and the organic solvent phase. Supersaturation is the force behind all these processes influencing the size and distribution of the NP population. The growth of the nanoparticles will terminate due to the combined effect of the polymer/drug dilution and steric hindrance of Pluronic F127 which deposits at the polymer core/water interface, affecting the aggregation dynamic, particle size, and the drug content of the NPs. Therefore, during the nanoprecipitation process, the type and ratio of solvent to non-solvent, as well as the polymer/drug/stabilizing agent concentrations, have to be carefully selected to achieve high efficacy of drug encapsulation, adequate particle size, and low polydispersity index ,29,30. OTransmission and Scanning Electron microscopes (TEM and SEM), respectively, were used to characterize the shape of the PLGA-PEG-PLGA NPs. The size of loaded PLGA-PEG-PLGA NPs prepared with 5 mg, 10 mg, and 15 mg Ponatinib has been measured in water dispersion using the Zetasizer Nano ZS . The particle size range of the NPs with increasing loading of Ponatinib was from 80 to 100 nm. The Z-average hydrodynamic diameter (Dh (nm) ± SD, n = 6) of each NPs’ group was as follows: 74.55 nm ± 28.74 for the PLGA-PEG-PLGA NPs loaded with 5 mg Ponatinib , 125 nm The Z-average hydrodynamic diameter (Dh (nm) ± SD) of the unloaded PLGA-PEG-PLGA NPs was 84.33 nm ± 13.83 , indicatThe surface charge of the PLGA-PEG-PLGA NPs (water dispersion) had been assessed to characterize more of its material properties; thus, its interaction properties with the biological system can be predicted. For that, the Zeta potential for the loaded PLGA-PEG-PLGA NPs had been measured by the Zetasizer Nano ZS , and the surface charge ((mV) ± SD, n = 5) of the particles showed a net positive charge of 12.3 mV ± 5.5; 15.2 mV ± 3.4 and 16.7 mV ± 2.5 for 15, 10 and 5 mg Ponatinib loaded PLGA-PEG-PLGA NPs.The Zeta potential for the unloaded PLGA-PEG-PLGA NPs demonstrated a net negative surface charge with an average of −2.66 ± 0.185 (mV) ± STD Dissolution rate experiments were performed using the dialysis method. In a phosphate buffer pH 7.4 pointed to a very slow dissolution rate from the prepared Ponatinib PLGA-PEG-PLGA NPs with no burst release except for the (sample A) prepared using 5 mg Ponatinib or 1:6 drug/polymer ratio (25% of the drug was released within the first 3 hours). For samples B and C, prepared using 10 and 15 mg Ponatinib (1:3 and 1:2 drug/polymer ratio), respectively, less than 10% released drug was determined within 24 h from all the samples . Results are demonstrated in The efficacy of loading and drug content increased with the increasing concentration of Ponatinib during preparation. Calculated values were 18 ± 3.3% (n = 6), 20.8 ± 2.1% (n = 6) and 21.8 ± 2.7% (n = 6) for 5 mg, 10 mg, and 15 mg Ponatinib; or 1:6; 1:3, and 1:2 drug to polymer ratio during the preparation of the NPs. Calculated drug content was 3%, 6.5%, and 10.5% for samples prepared with 1:6, 1:3, and 1:2 drug-to-polymer ratios, respectively.The survival rate of the zebrafish embryos at 72 h post-fertilizing (hpf) was calculated for the negative control (NC) which was untreated embryos kept in egg water and the treated groups of unloaded PLGA-PEG-PLGA NPs. First, we had investigated the effect of treating 72 hpf zebrafish embryos with Ponatinib (2.5 µm) on different cardiac function parameters. Cardiac function parameters measurements were examined on the zebrafish embryos’ two main blood arteries—(DA) dorsal aorta and (PCV) posterior cardinal vein—to examine the effect of Ponatinib treatment on the cardiovascular system of the zebrafish. Velocity, diameter, and pulse were measured using ZebraLab software. Cardiac parameters measurement was only possible on Ponatinib (2.5 μM) exposed embryos because of the severe effect of higher concentrations of Ponatinib on embryos’ viability. As demonstrated in We followed this with the investigation of (DA) and (PCV) vessel diameter, and blood flow velocity in the zebrafish embryos at 72 h -post fertilizing (hpf) treated with  unloaded PLGA-PEG-PLGA NPs. It was demonstrated that there was a significant reduction in the heartbeat of the group which was treated with unloaded PLGA-PEG-PLGA NPs (0.75 mg/mL) when compared to the negative control The dorsal aorta (DA) vessel diameter was seen to be enlarged in groups of  and the blood velocity was increased significantly in the unloaded PLGA-PEG-PLGA NPs (0.25 mg/mL) group compared to the negative control In the posterior cardinal vein (PCV) the vessel diameter showed to be enlarged in groups treated with unloaded PLGA-PEG-PLGA NPs , and also the blood velocity increased significantly in the unloaded PLGA-PEG-PLGA NPs (0.25 mg/mL) group A simple visual comparison of treated zebrafish embryos to controls at 24, 48, and 72 hpf was undertaken to explore Ponatinib’s teratogenic potential. Aristolochic acid (AA) (1 µM) was used as a positive control (PC) that induces cardiac failure per prior research ,32. PonaThe survival rate of the zebrafish embryos at 72 h post-fertilizing (hpf) was calculated for the negative control and the treated groups with three concentrations of loaded PLGA-PEG-PLGA NPs with Ponatinib: 3% of the drug content (sample A), 6.5% of drug content (sample B), and 10.5% of drug content (sample C). Based on these results, only high concentrations of loaded NPs were toxic to the embryos and the concentration of PLGA-PEG-PLGA (0.001 mg/mL) of samples (B and C) showed a similar survival rate to the negative control. Thus, this concentration is considered to be the optimum for performing the next experiments.The cardiac function was assessed by analyzing the heartbeat, the dorsal aorta (DA) and posterior cardinal vein (PCV) vessel diameter, and blood flow velocity.The heartbeat of groups treated with sample A NPs (0.005 and 0.0025 mg/mL), sample B NPs  sample C NPs (0.0025 and 0.001 mg/mL) were significantly reduced compared to the negative control, as shown in Blood velocity was significantly reduced in treated groups with sample C NPs at a concentration of (0.005 mg/mL) and slightly high in the treated group with sample C NPs at a concentration of (0.0025 mg/mL). The dorsal aorta (DA) vessel diameter demonstrated an enlargement in the groups treated with sample A NPs at a concentration of (0.005 and 0.0025 mg/mL), sample B NPs at a concentration of (0.0025 mg/mL), and sample C NPs at a concentration of (0.0025 and 0.001 mg/mL) as shown in In the posterior cardinal vein (PCV), the measured vessel diameters were significantly enlarged in all drug and NPs combinations, except for the group treated with sample C NPs (0.005 mg/mL), where the blood velocity was significantly reduced in Relying on these data, the concentration of NPs (0.001 mg/mL) of samples B and C manifested a non-toxic effect on treated groups; therefore, these concentrations would be used further in the xenograft experiments.K562 CML cell line was successfully transplanted into the 72 hpf zebrafish embryos. K562 CML cell line was also successfully transplanted when injected into the 48 hpf zebrafish embryos. On the same day of K562 cells injection, the 2 dpf xenograft embryos were exposed to PLGA-PEG-PLGA NPs (0.001 mg/mL) loaded with 10 mg Ponatinib. As shown in As shown in Since cancer is one of the main causes of death worldwide, many research studies are currently focusing on finding novel and efficient therapeutic tools to reduce side effects associated with conventional therapies for cancer . NanomedMoreover, nanomedicine could also support the cardio-oncology field which is an inter-disciplinary field of studying, detecting, and treating cardiovascular adverse effects associated with cancer therapies . AlthougIn the current study, the smart PLGA-PEG-PLGA NPs were synthesized by the nanoprecipitation method. The PLGA-PEG-PLGA NPs characteristics  had been investigated. The size of the unloaded PLGA-PEG-PLGA NPs was approximately 84.33 nm. Sulaiman et al. (2019) has shown that the PLGA-PEG-PLGA NPs size is in the range of 206 to 402 nm, Dimchevska et al., (2017) demonstrated that the size will vary depending upon the experimental conditions and polymer characteristics, and the most efficient way to optimize the formulation is to use experimental design for preparing PLGA-PEG-PLGA NPs ,40. SEM The cardiotoxicity of Ponatinib (2.5 μM) and the unloaded PLGA-PEG-PLGA NPs have been investigated to determine if this type of polymers would cause any cardiotoxicity or adverse effects. Our results demonstrated a significant effect of Ponatinib on reducing the velocity of aortic and PCV blood flow; these results came in agreement with a previous study conducted by Singh and coworkers . The zebBefore Ponatinib loading to NPs cardiotoxicity experiments we investigated the effect of treating zebrafish embryos with different concentrations of Ponatinib on their viability, tail flicking, and hatching rate at different time points, it has been shown that Ponatinib had the minimum effect on all measured parameters at a concentration of (2.5 μM). The dosing rationale that was tested for Ponatinib was based on the literature . IncreasThe smart NPs PLGA-PEG-PLGA loaded with Ponatinib  were used to reduce the drug’s toxic side effects, especially on the cardiovascular system. The loading of the drug was successfully performed, and this was indicated by the change in the surface charge of the PLGA-PEG-PLGA NPs from negative (−2.66 mV) to positive  charge for C, B, and A samples, respectively during the drug incorporation. Ponatinib surface charge is positive (30.86 mV) due to protonation of its on its terminal methylpiperazinyl nitrogen across water . This phThe cardiotoxicity of different concentrations of sample B and sample C PLGA-PEG-PLGA NPs was tested in zebrafish embryos before the investigation of their efficacy, as a less cardiotoxic therapeutic tool to treat CML. The concentration of 1, 0.75, 0.5, 0.25, 0.1, 0.025, 0.05, and 0.01 mg/mL NPs of all groups  had shown very clear toxicity based on demonstrated embryo survival rates which were the lowest associated with abnormal morphology.This was similar to the toxicity results of the treatment of Ponatinib drug only, as the embryos were deformed with heart edema and abnormal heart structure as well as for the absence of blood flow in the PCV and DA. This could be due to the burst release of Ponatinib which has been determined to cause cardiotoxicity. However, the lowest concentrations (0.005 and 0.0025 mg/mL) had shown a better effect but still, there were some observed abnormalities in the embryos, thus sample A PLGA-PEG-PLGA NPs were excluded and a lower concentration (0.001 mg/mL) from samples B and C PLGA-PEG-PLGA NPs were tested, and they demonstrated the best results in terms of survival rate, normal morphology, and cardiac output.Successfully, a zebrafish xenograft model has been generated to investigate the efficacy of those loaded with sample B and C PLGA-PEG-PLGA NPs, in reducing cardiotoxicity and as effective anti-cancer therapy to treat CML. This was achieved by transplanting the human K562 cell line into 2 dpf zebrafish embryos. This xenograft model has also been successfully generated and confirmed by the spread of the tumor cells to distal sites from the yolk sac throughout 6 days-post-injection, which is consistent with previous studies. Corkery et.al. (2011) has also used the K562 cells that were stained by the CM-Dil dye to give a red fluorescence color. These cells were then transplanted into the zebrafish embryos and the embryos were then kept for 1 h at 28 °C for a recovery period and this aided in enhancing the embryos’ survival rate . HoweverFinally, samples B and C PLGA-PEG-PLGA NPs (0.001 mg/mL) concentration were introduced to the injected zebrafish embryos (2 dpf) half an hour after the injection. The tumor cells are not reduced clearly over the 6 days after injection, this could be due to the long release time of Ponatinib from the PLGA-PEG-PLGA NPs.Possible limitations of this study includes that it being mostly dependent on zebrafish embryos that needed proper care and training for handling and that the xenograft model required an even a higher level of handling as the embryos are injured. The treated groups of the zebrafish embryos with the loaded PLGA-PEG-PLGA NPs were only observed till the ethical endpoint of 7-dpf,;thus, the effect of the loaded PLGA-PEG-PLGA NPs in reducing tumor cells was only observed for a few days despite the fact that Ponatinib release that could happen after a few days of the endpoint. For that, xenografted embryos need to be observed for a longer time, e.g., 10-dpf. Moreover, due to the lack of FTIR < X-ray and DSC studies, the paradox of the presence of burst release at the lowest drug concentration cannot be explained. Moreover, deducting background fluorescence per unit area of the fluorescence images would give better quantitative measurements.In summary, the zebrafish is a suitable animal model for investigating the cardiotoxicity associated with anti-cancer drugs such as TKIs, determining the optimum concentration of smart NPs with the least side effects, and generating a xenograft model of several cancer types.In this study, PLGA-PEG-PLGA NPs were synthesized to carry the TKIs drugs. These NPs have been shown to carry Ponatinib drugs (Samples B and C for a long time, allowing for longer circulation in the zebrafish body. Zebrafish animal model was used for testing the cardiotoxicity of a range of different concentrations of loaded and unloaded PLGA-PEG-PLGA NPs and the least concentrations were shown to be of low toxicity and enhanced survival rate. The concentrations of 0.1 and 0.05 mg/mL of the unloaded PLGA-PEG-PLGA NPs are the best in terms of low cardiotoxicity and high survival rate, while 0.001 mg/mL concentration of samples B or C PLGA-PEG-PLGA NPs has been shown to be the optimum concentration among the rest of the concentrations. Lastly, these loaded NPs have been exposed to the successfully generated CML xenograft zebrafish model, however, no obvious reduction in the tumor mass was seen, indicating the slow release of Ponatinib from PLGA-PEG-PLGA NPs.Generally, PLGA-PEG-PLGA NPs could be a good candidate for CML treatment, but their cellular internalization should be enhanced. This could be achieved by coating and labeling the surface of PLGA-PEG-PLGA NPs with specific ligands that are unique to CML cells.


Lactobacillus fermentum FXJCJ6-1, Lactobacillus brevis 173-1-2, and Lactobacillus reuteri CCFM1040. Our results showed that L. fermentum FXJCJ6-1 accumulated but did not use intracellular trehalose in a hypertonic environment, which enhanced its freeze-drying resistance. Furthermore, genes that could transport trehalose were identified in this bacterium. In addition, both the lactose addition and lactose culture improved the freeze-drying survival of the bacterium. Further studies revealed that the added lactose might exert its protective effect by attaching to the cell surface, whereas lactose culture acted by reducing extracellular polysaccharide production and promoting the binding of the protectant to the cell membrane. The different mechanisms of lactose and trehalose in enhancing the freeze-drying resistance of bacteria identified in this study will help to elucidate the anti-freeze-drying mechanisms of other sugars in subsequent investigations.Freeze-drying is a commonly used method in commercial preparations of lactic acid bacteria. However, some bacteria are killed during the freeze-drying process. To overcome this, trehalose and lactose are often used as protective agents. Moreover, high-density culture is an efficient way to grow bacterial strains but creates a hypertonic growth environment. We evaluated the effects of trehalose and lactose, as a primary carbon source or as an additive in fermentation, on the freeze-drying survival of  Lactobacillus preparations are lyophilized products prepared by vacuum freeze-drying in the presence of freeze-drying protectants. After freezing and drying, both of which are extreme processes, the bacteria are subjected to mechanical damage, solute damage, cell membrane damage, protein denaturation and inactivation, and DNA damage, which greatly reduce their survival rate [Lactic acid bacteria are a group of microorganisms that ferment carbohydrates to produce large amounts of lactic acid , and theval rate . TherefoLactobacillus plantarum during freeze-drying and storage [A common method for improving freeze-drying survival is the addition of freeze-drying protectants prior to freeze-drying, with sugars being the most common type of protectant. There are multiple possible mechanisms behind the anti-freeze-drying effect of sugars. The water replacement theory suggests that sugars displace water molecules and thus stabilize the structure of phospholipid cell membranes during drying. The glass transition theory states that sugars can lodge between the cell wall and the cell membrane and increase the glass transition temperature, thus mitigating mechanical damage due to the ice crystal formation in the bacterium . Therefo storage . A combi storage . In addi storage . HoweverOwing to the diverse mechanisms available to alleviate freeze-drying damage, there have been various studies on the addition of sugars, including extracellular polysaccharides, intracellular compatible solutes, intermembrane protectants, and the use of co-envelope materials to improve the freeze-drying survival of bacteria. The presence of carbohydrates on both sides of the bacterial membrane protects not only the bacterial membrane but also cytoplasmic proteins . The preA high-density culture is a fermentation method in which bacteria are cultivated using certain culture techniques and devices to significantly increase the bacterial density compared with conventional culture. This culture technique can thus increase the specific productivity of the target product, ultimately producing more bacteria at a lower cost. Under high-density culture conditions, a hypertonic but less inhibitory environment can be created via constant alkaline supplementation to control the pH of the medium and increase the density of the organisms ,13,14. ILactobacillus in high-density culture. The ability of the bacterium to metabolize the sugar, as well as a culture under hyper-versus hypotonic conditions, was used as a variable factor. Extracellular polysaccharides, cell membrane stability, and intracellular compatible solute accumulation were selected as study targets. The present study aimed to elucidate the mechanisms of the anti-freeze-drying effects of sugars. In this study, we focused on the effects of different sugar additions during bacterial growth on their freeze-drying resistance rather than as freeze-drying protectants and revealed the possible mechanisms. We investigated the effect of the addition of trehalose and lactose, both as a carbon source culture and as an additive, on the freeze-drying survival of Lactobacillus fermentum FXJCJ6-1, Lactobacillus reuteri CCFM1040, and Lactobacillus brevis 173-1-2 were obtained from the Culture Collection of Food Microbiology (CCFM) at Jiangnan University .The strains were cultured on MRS plates, and the plates were incubated upside down in a constant temperature incubator at 37 °C for 24–36 h. A single colony was selected from the growing colonies and incubated in MRS liquid medium at 37 °C for 12 h. The seed solution was added to fresh the MRS liquid medium with a 4% inoculum and incubated at 37 °C for 12 h. The activated seed solution was obtained after two to three repetitions.L. fermentum FXJCJ6-1 at a constant pH (6.0) batch culture contained 40 g/L yeast extract, 108 g/L glucose, 0.175 g/L MgSO4, 0.175 g/L MnSO4, and 1 mL/L Tween 80. The medium for L. reuteri CCFM1040 at a constant pH (5.0) contained 40 g/L yeast extract, 112 g/L glucose, 0.175 g/L MgSO4, 0.175 g/L MnSO4, and 1 mL/L Tween 80. The medium for L. brevis 173-1-2 at a constant pH (5.0) contained 40 g/L yeast extract, 108 g/L glucose, 0.125 g/L MgSO4, 0.125 g/L MnSO4, and 1 mL/L Tween 80. Based on our preliminary studies, the medium compositions for the strains were designed as follows. The medium for Lactobacillus strains in different carbon sources, glucose was replaced with lactose, sucrose, or trehalose according to the glucose content in the corresponding basal medium for each strain.For the culture mediums of the three g and 4 °C for 20 min. The cells were mixed thoroughly with the protectant solution at a mass ratio of 1:1. Next, 1 mL of the mixture was pipetted into an 8 mL freeze-drying glass bottle (id = 22 mm) for freeze-drying. The protective agent solution consisted of oligoisomaltose 75 g/L and collagen 25 g/L.The fermentation broth was collected, and the supernatant was removed by centrifugation at 8000× Freeze drying was carried out using a Telstar freeze dryer . The freeze-drying process was set to the following. The temperature of the laminate was controlled to drop to −50 °C from room temperature within 1 h and to maintain this temperature for 4 h. Then, the vacuum degree was controlled, and the temperature of the freeze dryer shelf was adjusted to −30 °C within 1.3 h, which was maintained under a 0.2 mbar vacuum degree for 30 h. Lastly, the temperature of the freeze dryer shelf was controlled for 1 h, raised to 25 °C, and maintained for 20 h under the condition of a 20 μbar vacuum.The number of viable bacteria (CFU/mL) in each sample before freeze-drying was determined. Next, sterile saline was added to the freeze-dried powder of the same mass before freeze-drying. Finally, the number of viable bacteria (CFU/mL) in the samples after freeze-drying was determined using the plate colony counting method to calculate the freeze-drying survival rate, expressed as:NA—number of live bacteria in the sample before freeze-drying; NB—number of live bacteria in the sample after freeze-drying.g for 10 min. The supernatant was removed, and the precipitate was washed three times with an isotonic phosphate-buffered saline (PBS) solution to obtain the cells to be examined. Next, 0.75 mL of glutaraldehyde fixative for electron microscopy was added to the centrifuge tube, and the tube was left for 2 h at 4 °C. After centrifugation to remove the supernatant, the samples were rinsed thrice with a PBS solution for 30 min. The samples were then precipitated with ethanol and dehydrated with a 1:1 (v/v) mixture of anhydrous ethanol, tert-butanol, and pure tert-butanol solution once for 15 min each. Finally, the samples were dried using a vacuum concentrator. For scanning electron microscopy, the sample was attached using conductive tape to the microscope stage. The surface of the sample was plated with a gold film with a thickness of 100–150 Å. The appropriate magnification and field of view were then selected to observe the morphology of the bacterium.The fermentation broth (1 mL) was centrifuged at 8000× g for 15 min. The supernatant was removed, and the sample was washed thrice with an isotonic PBS solution. The extractant (acetonitrile: methanol: water = 2:2:1) was added to the sample and shaken well to suspend the slime in the extractant. The mixture was repeatedly freeze–thawed in liquid nitrogen three times to break the bacterium. The metabolites were dissolved, and the extract was collected. The extraction procedure was repeated. The metabolites were mixed twice and transferred to a centrifuge tube. Finally, the sample was blown dry using a vacuum concentrator and then stored at −80 °C.The fermentation broth was centrifuged at 8000× The chromatographic conditions for the detection of sugars were as follows. Waters-600 HPLC was used with a Sugarpak I column at a column temperature of 85 °C, using the evaporative light scattering detection method. The mobile phase was ultrapure water, and the flow rate was 0.4 mL/min.g for 15 min. Next, the supernatant was removed, and 5% Trichloroacetic acid solution  was added to precipitate the protein. The mixture was stored at 4 °C for 30 min and then centrifuged at 10,000× g for 15 min. The supernatant was removed, and anhydrous ethanol was added to the supernatant  for alcoholic sedimentation at 4 °C overnight. After centrifugation at 10,000× g for 15 min, a flocculent precipitate was obtained, collected, dissolved in an equal volume of ultrapure water, and packed into a dialysis bag (8000 D). The water was changed once at 8 h. After 48 h of ultrapure water dialysis, the precipitate was removed and lyophilized to obtain extracellular polysaccharides.The bacterial solution was collected, sonicated at 25 °C for 10 min (500 w), and centrifuged at 10,000× 620 was determined, and the extracellular polysaccharide content was calculated according to the standard curve.Anthrone (0.2 g) was dissolved in 100 mL of 80% concentrated sulfuric acid. The above polysaccharide precipitate was dissolved in ultrapure water and diluted with water to 1 mL. Next, 4 mL of sulfuric acid-anthrone solution was added. The mixture was then removed from the boiling water bath for 10 min and cooled to room temperature. The ODp < 0.05.All experiments were repeated three times, and the data are presented as the mean ± standard deviation. Experimental data were plotted using Origin 9.1, and one-way ANOVA (Duncan’s test) was performed using SPSS . Statistical significance was set at Lactobacillus strains were cultured at high density using different sugars as the carbon source and then were lyophilized to determine the number of viable bacteria and the freeze-drying survival rate % compared to the glucose group (p < 0.05). Moreover, the addition of lactose significantly increased the freeze-drying survival of L. reuteri CCFM1040 and L. brevis 173-1-2 compared to the glucose group (p < 0.05). As shown in L. fermentum FXJCJ6-1 could not utilize trehalose, and L. brevis 173-1-2 could not utilize lactose. Therefore, the addition of non-metabolizable sugars may have beneficial effects on freeze-drying resistance. Thus, we will focus on L. fermentum FXJCJ6-1 and L. brevis 173-1-2 to study their freeze-drying resistance with the addition of trehalose or lactose, respectively.The addition of trehalose significantly increased the freeze-drying survival of L. fermentum FXJCJ6-1, but this strain was unable to utilize trehalose as a carbon source. Combined with the fact that bacteria have been found to take up compatible solutes from outside to counteract adverse stress in a hypertonic environment, several studies have shown that the accumulation of carbohydrates in their cells could help bacteria resist desiccation stress [L. fermentum FXJCJ6-1 when cultured in a hypertonic environment was significantly higher than that of a hypotonic environment (p < 0.05). Moreover, only the addition of trehalose in the hypertonic environment could significantly increase the freeze-drying survival rate of the strain (p < 0.05). Therefore, it was speculated that L. fermentum FXJCJ6-1 intracellularly accumulated trehalose as a compatible solute in the hypertonic environment to improve resistance to adversity.The above results show that the addition of trehalose greatly enhanced the freeze-drying survival of n stress . High inn stress ,16. It hn stress . We specL. fermentum FXJCJ6-1 accumulates trehalose intracellularly to improve its resistance during freeze-drying, we examined the intracellular and extracellular trehalose content of L. fermentum FXJCJ6-1 when grown under hypertonic and hypotonic environments . Therefore, we speculate that this gene is similar in function to treB and capable of transporting trehalose into the cytosol. In contrast, no genes related to trehalose transport were identified in L. reuteri.To test the conjecture that ronments . The res of treB   can lead to increased resistance to freeze-drying . TherefoL. reuteri CCFM1040 and L. fermentum FXJCJ6-1 was significantly reduced in the medium with lactose as the sole carbon source (p < 0.05). Extracellular polysaccharides contribute to the resistance of an organism to stress [Previous studies have shown that changes in culture conditions can lead to changes in extracellular polysaccharide production by bacteria . As showo stress . Howevero stress . When tho stress . As a reLactobacillus, mainly in the following aspects: first, the addition of different sugars to the culture media will affect the morphology and size of bacteria; second, in a hypertonic environment, some strains resistant to high osmotic pressure are able to transfer compatible solutes, such as trehalose and sucrose, into the cell, thereby regulating the osmotic pressure balance inside and outside the cell and improving the resistance of the strain itself; third, sugars in the medium can affect the extracellular polysaccharide production of lactic acid bacteria. The reduction in polysaccharides increases the efficacy of the protective agent on the bacterium. In this study, we investigated whether the metabolism of trehalose and lactose by Lactobacillus strains influenced their freeze-drying survival rate after incubation under hypertonic conditions. Our hypotheses were as follows: (1) strains that could not metabolize trehalose and lactose had increased freeze-drying survival rates after the addition of trehalose and lactose, and they could intracellularly accumulate in trehalose and lactose under hypertonic conditions, leading to an increase in their freeze-drying survival rate; (2) strains that could metabolize lactose had increased freeze-drying survival rates after incubation with lactose, either because of changes in morphology and extracellular polysaccharide production during lactose fermentation or via the intracellular accumulation of lactose, leading to increased freeze-drying survival rates.Sugars are an important component of microbial media, not only as a carbon source to provide nutrients for cell proliferation but also as they influence the stress resistance of microbes. Different sugars affect the freeze-drying resistance of L. fermentum FXJCJ6-1 to intracellularly accumulate trehalose to resist freeze-drying stress. The freeze-drying survival of L. fermentum FXJCJ6-1 increased by (27.53 ± 1.36)% after trehalose was added to the medium. This finding is similar to those of other studies [Lactobacillus were inhibited in a hypertonic environment and that cells translocated compatible solutes from the environment to resist environmental stresses [L. acidophilus is a trehalose-transporting protein [L. fermentum FXJCJ6-1 also has the ability to transport trehalose. We also investigated the freeze-drying resistance mechanism and found that cultures under hypertonic conditions induced  studies . Song et studies . MD Angestresses . When osstresses . Consist protein , we founWhen a trehalose-transporting protein is present in a bacterium, hypertonic conditions may induce the transport of trehalose into the cytosol, thereby increasing its resistance to stress. Therefore, further investigation of the mechanisms by which environmental conditions affect trehalose transport can be carried out using this gene. Moreover, other strains with trehalose transport systems can be predicted for the more effective examination of the anti-freeze-drying effect of trehalose.L. fermentum FXJCJ6-1 and L. reuteri CCFM1040 grown in a lactose culture medium, whereas their freeze-drying survival increased by (24.92 ± 1.74)% and (15.68 ± 2.61)%, respectively. Several studies have found that extracellular polysaccharides are key substances for improving the freeze-drying survival of Lactobacillus [Colwellia psychrerythraea, incubated the bacterium with the exopolysaccharides as a protective agent, and lyophilized the culture [In addition, we found a significant reduction in the extracellular polysaccharide production for bacillus ,29,30. Ebacillus . Joseph  culture . Compare culture . HoweverLactobacillus stains. The addition of trehalose and lactose during bacterial growth may significantly increase their freeze-drying survival. Hypertonic conditions were beneficial for the transport of trehalose into the bacterial cell, thereby improving the freeze-drying resistance of L. fermentum FXJCJ6-1. Lactose as a carbon source also significantly improved the freeze-drying survival of L. fermentum FXJCJ6-1 (A) and L. reuteri CCFM1040. One possible reason may be the decrease in the production of extracellular polysaccharides after incubation with lactose, which was conducive to the protective effect of the freeze-drying protectant. Overall, our results provide an alternative strategy to improve the freeze-drying survival of LAB strains, as well as theoretical support for the addition of suitable sugars in the culture process.Overall, we found significant differences between the efficacies of trehalose and lactose in improving the freeze-drying survival of


Aspalathus linearis (Burman f.) R. Dahlgren (known as rooibos tea) exhibits a wide range of activities beneficial for cardio-metabolic health. Thus, the present study aims to investigate unfermented and fermented rooibos extracts and their constituents for the ability to trap MGO and GO. The individual compounds identified in extracts were tested for the capability to inhibit AGEs (with MGO or GO as a glycation agent). Ultra-high-performance liquid chromatography coupled with an electrospray ionization mass spectrometer (UHPLC–ESI–MS) was used to investigate α-DCs’ trapping capacities. To evaluate the antiglycation activity, fluorescence measurement was used. The extract from the unfermented rooibos showed a higher ability to capture MGO/GO and inhibit AGE formation than did the extract from fermented rooibos, and this effect was attributed to a higher content of dihydrochalcones. The compounds detected in the extracts, such as aspalathin, nothofagin, vitexin, isovitexin, and eriodictyol, as well as structurally related phloretin and phloroglucinol (formed by the biotransformation of certain flavonoids), trapped MGO, and some also trapped GO. AGE formation was inhibited the most by isovitexin. However, it was the high content of aspalathin and its higher efficiency than that of metformin that determined the antiglycation and trapping properties of green rooibos. Therefore, A. linearis, in addition to other health benefits, could potentially be used as an α-DC trapping agent and AGE inhibitor.The excessive dietary intake of simple sugars and abnormal metabolism in certain diseases contribute to the increased production of α-dicarbonyls (α-DCs), such as methylglyoxal (MGO) and glyoxal (GO), the main precursors of the formation of advanced glycation end products (AGEs). AGEs play a vital role, for example, in the development of cardiovascular diseases and diabetes.  Nowadays, it is well established that diet can significantly affect the metabolic health of the human body. In recent years, dietary habits have evolved all over the world, and the consumption of simple carbohydrates in the diet of the average person in developed countries has increased dramatically . The incHowever, it is assumed that α-DC activity exceeds that of the reduction of sugars by up to several hundred times. The process of non-enzymatic glycation occurs under physiological conditions in the body . NeverthAspalathus linearis (Burman f.) R. Dahlgren, also known as rooibos or redbush tea, is a popular plant raw material widely used around the world, and its beneficial health properties, especially in the context of cardiometabolic health, are fairly well recognized −. Another three chromatographic peaks at m/z 269 were recognized as isomers of di-MGO-phloroglucinol. Their daughter ions with m/z 179 suggested the loss of one molecule of methylglyoxal and one molecule of water [M–72–18–H]−, while the ions with m/z 125 corresponded to the precursor ion, indicating the dissociation of two MGO molecules [M–144–H]−. The remaining two chromatographic peaks were annotated as tri-MGO-phloroglucinol isomers because the m/z of ions matched that of phloroglucinol increased by three methylglyoxal molecules and was 341. A similar decomposition pattern to that of di-MGO-phloroglucinol was observed herein, as ions at m/z 251 corresponded to tri-MGO-phloroglucinol with a loss of methylglyoxal and water [M–72–18–H]–. In addition, a characteristic ion at m/z 125 was also evident, representing the deprotonated phloroglucinol ion and implying the detachment of three methylglyoxal molecules [M–216–H]–. In the reaction of phloroglucinol with glyoxal, only two additional peaks appeared on the chromatogram after one hour. The first one was assigned as di-GO-phloroglucinol since its pseudo-molecular ion was at m/z 241, which is equivalent to the phloroglucinol precursor increased by 116 Da, meaning two GO molecules. Meanwhile, the second peak was designated as mono-GO-phloroglucinol since its m/z of 183 analogously corresponded to the attachment by phloroglucinol of one glyoxal molecule (58 Da). Mass spectra of the adducts formed by phloroglucinol and MGO/GO are shown in In the 1 h reaction of phloroglucinol with methylglyoxal, the formation of as many as seven new peaks on the chromatogram was observed. Two of these peaks with Fucus vesiculosus L., obtaining results coinciding with our results. The authors of another publication describing the structural requirements for flavonoids to trap methylglyoxal stated that the A-ring is essential for MGO uptake, and the hydroxyl group at C-5 of the A-ring is strongly supportive of the methylglyoxal trapping capacity − corresponded to the phloretin deprotonated ion (m/z 273) increased by 144 Da (two MGO). The remaining formed adduct had an ion m/z of 345 and was designated as mono-MGO-phloretin since its mass corresponded to that of phloretin after the attachment of one MGO molecule. In the one-hour reaction of phloretin with glyoxal, the formation of a single monoadduct was observed with m/z equal to 331. The ion of the phloretin m/z 273 corresponded to the mass of the mono-GO-phloretin adduct minus the mass of one molecule of glyoxal [M–58–H]−. This is consistent with previous reports of phloretin’s ability to trap MGO and GO and confirms that the experimental conditions provided the potential for adduct formation. Mass spectra of the adducts formed by phloretin and MGO/GO are shown in Dihydrochalcone with known trapping properties served as a reference compound in the experiment; phloretin produced three new chromatographic peaks after 1 h of incubation with methylglyoxal. Two of these peaks, whose pseudo-molecular masses were equal to m/z ion mass of 523 corresponding to that of the aspalathin ion plus one molecule of methylglyoxal. In addition, the degradation profile corresponded to that observed for aspalathin in the extract, with the presence of daughter ions at m/z 505 [M–18–H]– and 415 [M–36–72–H]– reflecting the loss of one water molecule or two water molecules and one methylglyoxal molecule, respectively (described above). The presence of as many as four mono-MGO-aspalathin adducts was identified in GRE; two of them were identical to the aspalathin standard and the other two appeared on the chromatogram at around 26 and 27 min. These were probably isomeric forms, which were formed by different ways of attaching MGO to the phloroglucinol ring of aspalathin at the C-5′ position and the -OH group at the 4′ or 6′ position or the presence of an aspalathin isomer in the extract . To the best of our knowledge, this is the first time that the trapping ability of methylglyoxal has been described for nothofagin. Mass spectra of the adducts formed by nothofagin and MGO are shown in Since we did not use a nothofagin standard, observations were made only for the nothofagin contained in the green rooibos extract. In the reaction with methylglyoxal, four new chromatographic peaks were formed, labeled as mono-MGO isomers of nothofagin. Their C-glycosylated flavones vitexin and isovitexin in a 1 h reaction with methylglyoxal produced two new chromatographic peaks, each with [M − H]− at 503, suggesting that under the experimental conditions two mono-adducts each were formed for vitexin and isovitexin. These deprotonated ions corresponded to a vitexin and isovitexin precursor ion mass of 431 plus one molecule of methylglyoxal (72 Da). The decay spectrum revealed the presence of ions 413 and 311 formed by the loss of fragments with 90 Da and 120 Da, respectively, from the cross-ring cleavages of the hexose unit. This indicates that in the cleavage, the glucose moiety is degraded first rather than the heterocyclic structure of the methylglyoxal adduct. Mass spectra of the adducts formed by vitexin and isovitexin and MGO/GO are shown in The Previous studies on vitexin and isovitexin in MGO trapping reported inconsistent results. A study by Peng et al.  showed tm/z 489 was equivalent to the mass of the vitexin precursor ion (m/z 431) increased by 58 Da, meaning one molecule of glyoxal.In the reaction of vitexin with glyoxal, two new peaks appeared on the chromatogram after one hour. Both were assigned as mono-GO-vitexin since their deprotonated ion at In this experiment, isovitexin was not observed to form adducts after incubation with glyoxal. Both vitexin and isovitexin have one position each in the A-ring where the dicarbonyl MGO/GO can be attached; these are the C-6 or C-8 sites, respectively. If, as in the case of vitexin, there is a C-6 carbon, then two adducts can be formed with an -OH group at C-5 or C-7 with two forms of hemiketal or hemiacetal for each version. For isovitexin with an unsubstituted C-8 position, adducts are formed only with an -OH group at C-7 and again with two forms of hemiketal and hemiacetal. Therefore, isovitexin can have only two mono-adducts, while vitexin hypothetically can produce as many as four. Although it is most likely that only two are preferentially predominant, we do not actually know which. Isovitexin did not produce adducts with GO, so the C-8 position appears to be less favored, most likely due to differences in the C-8 surroundings and substituents at adjacent positions (-OH group free or in the C-ring heterocyclic system). As indicated, these reactions lead to the formation of at least several different stereoisomers of adducts of specific compounds with α-DCs, and some of them are preferentially formed due to their favorable electron configuration. At this stage of research, however, we are not able to determine their chemical structures specifically. For this purpose, further structural studies are necessary.m/z 359, which corresponds to the ion of the precursor m/z 287 enlarged by 72 Da (one molecule of MGO). For the reaction of eriodictyol with glyoxal, two additional peaks appeared on the chromatogram after 1 h of incubation. Both of these peaks were identified as mono-GO-eriodictyol . In A. linearis, C-glycosides of eriodictyol have been identified; according to the literature, glucose is substituted into the eriodictyol in a manner similar to that observed in the C-glycosides of apigenin and luteolin (at the C-6 or C-8 position of the A-ring). We did not have standards for specific eriodictyol-C-glycosides, so we used aglycone; however, based on the results for vitexin and isovitexin, it can be assumed that the eriodictyol-C-glycosides found in rooibos may also at least partially possess MGO- and GO-trapping properties. Further studies are needed for confirmation. Mass spectra of the adducts formed by eriodictyol and MGO/GO are shown in After the incubation of MGO with the only representative flavanones, i.e., eriodictyol, four additional chromatographic peaks were noted on the chromatogram labeled as mono-MGO-eriodictyol. Their pseudo-molecular ions were at Although three different subgroups of flavonoids—dihydrochalcones , flavones (vitexin and isovitexin) and flavanones (eriodictyol)—show different C-ring structures, all can effectively capture methylglyoxal, indicating that the C-ring of flavonoids may not play an important role in trapping MGO. By contrast, for nothofagin and isovitexin, we did not observe the ability to form adducts with glyoxal, although the two compounds have a different C-ring structure: Nothofagin as a dihydrochalcone has an open C-ring, while isovitexin as a flavone has a C-ring in closed form.The structure of the B-ring does not seem to play a key role in the ability to trap α-dicarbonyls. In our test, both aspalathin with two -OH groups and phloretin and nothofagin possessing only one -OH group in the B-ring exhibited the ability to trap methylglyoxal. However, only phloretin was able to form diadducts with MGO, undoubtedly influenced by the fact that it does not have a substituted C-glucose in the A-ring. The importance of the A-ring structure of flavonoids was described above.In summary, A. linearis extracts were rich in dihydrochalcones, flavanones, flavones, and flavonols. However, green rooibos extract had several times more flavonoids—especially aspalathin and nothofagin—than red rooibos extract. In the antiglycation assay, isovitexin demonstrated the highest activity in both glycation models with MGO and GO, and its activity was statistically significantly higher than that of aminoguanidine, a compound considered one of the most potent inhibitors of non-enzymatic glycation. Only green rooibos extract inhibited the glycation process at a level comparable to isolated aspalathin , which suggests that it is aspalathin as the main component found in GR and GRE in the greatest amounts that determines the antiglycation activity. Indeed, red rooibos did not display the ability to inhibit protein glycation in any of the models. Some compounds tested, despite their ability to trap GO, showed no activity against glyoxal-induced glycation inhibition . This was likely influenced by factors such as pH, reaction time, process temperature, and the fact that glyoxal is present in hydrated forms in solution (described in detail below). Others, despite their lack of GO-trapping ability, were able to inhibit the glyoxal-triggered glycation process, perhaps due to the strong antioxidant properties that are a component of the antiglycation effect. This study revealed that under the experimental conditions, all tested compounds demonstrated activity towards the direct trapping of methylglyoxal, while only aspalathin, vitexin, and eriodictyol exhibited the ability to trap glyoxal. Phloretin and phloroglucinol used as reference compounds showed trapping activity against both α-dicarbonyls. No MGO or GO adducts were observed for RRE, which is probably due to low concentrations of flavonoids in the extract, compared to GRE (320.2 μM/L vs. 1443.1 μM/L). In GRE, only dihydrochalcones were a source of MGO  and GO  adducts.It is known that non-enzymatic antiglycation activity usually results from a number of different mechanisms of reaction. The literature reports that components of the inhibitory effect on the glycation process include antioxidant activity, the ability to chelate transition metals, and the capacity of compounds to trap α-DCs . FrequenC-glycosides of flavonoids such as aspalathin and nothofagin present in A. linearis, which are metabolized in the large intestine, may exert local beneficial anti-AGE and anti-α-dicarbonyl effects in conditions such as irritable bowel syndrome. Bacterial metabolites that act deleteriously in excess on the digestive system and cause unpleasant symptoms in the form of irritation play a major role in these conditions [An important aspect that should be considered is the fact that aspalathin, despite its high antiglycation as well as trapping properties, has an extremely low level of bioavailability—at less than 1% . Howevernditions . One sucnditions .Dried, crushed leaves and apical parts of shoots of Aspalathus linearis (Burm. f.) R. Dahlgren plantations in South Africa were used to prepare infusions and hydroethanolic extracts. Two types of raw materials were used: unfermented  and fermented  purchased from the Polish tea manufacturer Oxalis .v/v) for 15 min. The DER was 1:100. An ultrasonic bath  at 40 °C was used for hydroethanolic extraction. After 15 min, the extracts were centrifuged and then filtered using 0.22 μm diameter Durapore filters into vials. The filtrates were used for phytochemical analysis and in vitro tests. Hydroethanolic extracts from GR and RR were designated GRE and RRE, while infusions were designated GRI and RRI, respectively.Before the preparation of the extracts, both types of raw material were finely ground, each for 5 min, using an IKA A11B analytical mill . Then, 0.25 g of the powdered plant material was weighed on an analytical balance, placed in appropriately labeled volumetric flasks, and extracted with 25 mL of boiling water (infusions) or a mixture of water and ethanol  were obtained from Chempur . Water used in the study was deionized. The stock solutions of standards for in vitro assays were prepared by dissolving the reference compound in 5 mL of a suitable solvent, filtered through hydrophilic Millex Syringe Filters Durapore 0.22 μm  and stored at −20 °C.Methylglyoxal , glyoxal , methanol (HPLC grade), acetonitrile (HPLC gradient grade and LC-MS grade), water (LC-MS grade), bovine serum albumin, DMSO, 98–100% formic acid, phloroglucinol (CAS No. 108-73-6), phloretin (CAS No. 60-82-2), eriodictyol (CAS No. 552-58-9), aspalathin (CAS No. 6027-43-6), aminoguanidine hydrochloride (CAS No. 16139-18-7), and metformin hydrochloride (CAS No. 1115-70-4) were purchased from Merck-Sigma-Aldrich . Vitexin (CAS No. 3681-93-4), isovitexin (CAS No. 38953-85-4), hyperoside (CAS No. 482-36-0), isoquercitrin (CAS No. 482-35-9), rutin (CAS No. 153-18-4), eriodictyol-7-O-β-glucoside (CAS No. 38965-51-4), apigenin (CAS No. 520-36-5), luteolin (CAS No. 491-70-3), and chrysoeriol (CAS No. 491-71-4) were purchased from Extrasynthese . NaCl, KCl, Nav/v), filtered through hydrophilic Millex Syringe Filters, Durapore 0.22 μm  and stored at −20 °C.Stock solutions (1 mg/mL) for quantitative and semiquantitative analysis were made by dissolving 5 mg of flavonoid in 5 mL of methanol. Working standard solutions in the range of 10–250 g/mL (6 measurement points for each pattern) were prepared by mixing with 50% aq. methanol (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B). The following gradient mobile phase program at a flow rate of 0.3 mL/min was used: 0–12 min, 97–65% A in B; 12–14 min, 65% A in B; 14–17 min, 65–20% A in B; 17–19 min, 20% A in B. Then, the system was returned to the initial settings and washed with 97% A in B until the system was stabilized before the next analysis. Negative ion mode (ESI−) was used for data acquisition. Nitrogen was used as a nebulizing gas at 210 °C temperature, 2.0 bar pressure, and 0.8 L/min flow rate. For internal calibration, sodium formate clusters (10 mM) were used. The injection volume was 2.5 μL. Additional operating conditions of the mass spectrometer were as follows: the capillary voltage was set at 5 kV, the collisional energy was 8.0 eV, and for the MS2 mode, it was 40 eV. The data processing was carried out using Compass Data Analysis software . The same method was used to analyze the formation of MGO and GO adducts.Phytochemical analysis of extracts was conducted using the Thermo Scientific Dionex UltiMate 3000 UHPLC system  incorporated with Compact ESI-QTOF-MS , quaternary pump (LPG-3400D), and UltiMate 3000 RS autosampler (WPS-3000). Compounds were separated on a Kinetex C18 column   and a temperature-controlled column compartment (TCC-3000) was used to maintain its temperature at 40 °C. Mobile phases consisted of 0.1%  and D, 1.5% formic acid in acetonitrile (v/v). HPLC gradient was as follows: 10%, 25%, 65%, 80% (D in C) at time points of 0-30-33-50 min. The flow rate was 0.9 mL/min, and the injection volume was 20 μL. The column was operated at 20 °C. The spectral measurements were made in the wavelength range 200–600 nm in steps of 2 nm. Dihydrochalcones and flavanones were analyzed at 280 nm, while flavones and flavonols were analyzed at 360 nm.HPLC–DAD analysis was performed on a Smartline system  with a pump (Managare 5000), dynamic mixing chamber (V7119-1), DAD 2800 detector, manual 6-port 2-channel injection valve (A1366), and column thermostat (Jetstream Plus). Data were processed using EuroChrom for Windows Basic Edition V3.05 (V7568-5). The separation was carried out on a Hypersil GOLD C18 column  with a C18 precolumn  . The following eluents were used: C, 1.5% formic acid in water  used in the calculations was 0.999 to 0.9999.Flavonoid content  was determined using the external standard method from the areas of the corresponding peaks. Dihydrochalcones, flavanones, flavones, flavonols, and total flavonoids were calculated by summing the content of compounds from these (sub)groups. Nothofagin was calculated as an aspalathin equivalent. Similarly, orientin and isoorientin were expressed as vitexin and isovitexin, and eriodictyol-C-glucosides were expressed as eriodictyol-7-O-β-glucoside. The concentrations of other flavonoids were quantified using their corresponding standards. Mean content and standard deviations were calculated from five independent measurements. We also converted the obtained results into micromolar concentrations.m/v) sodium azide, which prevented microorganism growth in a test tube. Compounds investigated for antiglycation activity were added at a final concentration of 1.5 mM. Hydroethanolic extracts (DER 1:400) were added at a volume of 500 µL. Then, the reaction solution was incubated in simulated physiological conditions at 37 °C, shaken at 50 revolutions per minute for 7 days in closed vials secured with parafilm tape, and kept away from sunlight.The glycation model was created based on a slightly modified published method . In the ex) and 460 nm for emission (λem). Data processing was carried out using Gen5 Software . The measurements from three experiments were all performed in triplicate, and the percent inhibition of AGE formation was calculated using the following equation:0 is the mean fluorescence intensity of the blank sample and FI1 is the mean fluorescence intensity of the tested sample.The fluorescent intensity of α-DC-mediated AGEs formed during incubation was analyzed using a Synergy HTX Multi-Mode Microplate Reader  at a wavelength of 360 nm for excitation (λA. linearis hydroethanolic extract (DER 1:400) and 0.1 M PBS (pH 7.4 was determined immediately before use) at 37 °C and shaken at 50 rpm for 1 h. The reaction was stopped by adding 2.5 μL of glacial acetic acid and transferring Eppendorf tubes with the collected samples to an ice water bath. Next, the samples were carefully filtered through hydrophilic Millex Syringe Filters  and analyzed using UHPLC–ESI–MS . Data were analyzed using the Shapiro–Wilk test to assess the normality of distribution, followed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test using the GraphPad Prism 9 software, and Dicarbonyls are ubiquitous in our bodies since they are metabolic intermediates and can be generated from glycolysis and lipid peroxidation. Reducing their concentration in the human body can be achieved through diet—primarily by eliminating excess simple sugars from the diet. However, since in some pathological conditions their overproduction is not only related to diet, attempts are also being made to reduce their concentration in the body through the use of MGO- and GO-trapping compounds, among others.A. linearis can trap MGO and GO and inhibit the glycation process induced by α-DCs. Further studies using in vivo models are needed to confirm and expand our results.This study demonstrated that in addition to the many health properties attributed to green and red rooibos, this plant material may also be considered as a potential adjunctive anti-α-dicarbonyl agent preventing the onset and development of glycation-related conditions. Since there are currently no pharmacological strategies to decrease α-DC levels as an early preventive measure against AGE-induced diseases, it is important to search for adjunctive therapies. Our study indicates that green rooibos, probably due to its high concentration of aspalathin and nothofagin, exhibits MGO- and GO-trapping activity and antiglycation potential, unlike red rooibos. However, compounds present in both unfermented and fermented


Background: Acute appendicitis is the leading cause of acute abdominal pain that requires immediate intervention. Nonetheless, during COVID-19, hospital visits decreased as a result of serious COVID-19 concerns at that time, resulting in a decreased number of diagnosed cases with acute appendicitis due to COVID-19 restriction issues.Objectives: To report the percentage numbers, characteristics, applied management, and outcomes of patients with acute appendicitis during the COVID-19 pandemic and compare them to pre-COVID-19 cases.Methods: A retrospective cohort study included all patients with acute appendicitis in the determined periods “pre-COVID-19” and “during COVID-19“ at King Abdul-Aziz Medical City, Academic Tertiary Center, Jeddah, Saudi Arabia. Mean and standard deviation were used, while categorical data were reported as frequencies and percentages. Variables were analyzed by the Chi-squared test, Fisher’s exact test, and Mann-Whitney test as appropriate.Results: A total of 298 patients were included. The period of the pre-COVID-19 pandemic had 161 (54%) patients, while 137 (46%) were identified during COVID-19. The number of laparoscopic appendectomies performed during COVID-19 was less than the pre-COVID-19 pandemic of 96 cases (70.1%) vs 133 cases (82.6%) (P=0.0106). Uncomplicated appendicitis was the most commonly reported type of appendicitis in both periods: 113 (82.5%) during COVID-19 vs 135 (83.9%) pre-COVID-19, (P=0.7526). Furthermore, the number of patients who presented to the ER between 24 and 48 hours after the onset of symptoms was similar before and during the pandemic: 111 (68.9%) vs 89 (65%).Conclusion: Overall, we conclude that during the COVID-19 period, there was a reduction in the number of patients presenting with acute appendicitis and a lower chance of undergoing laparoscopic appendectomy due to COVID-19 restrictions. There was also an increase in perforated appendicitis and a decrease in gangrenous appendicitis. Acute appendicitis is the most common cause of acute abdominal pain that requires surgical intervention . It is dOn March 11, 2020, COVID-19 has been globally declared a pandemic health issue by the World Health Organization (WHO) . It had Nevertheless, after almost two years since the start of the first case of COVID-19 in the world, there is a lack of studies discussing the effects of the COVID-19 pandemic on acute appendicitis management and outcomes, especially in Saudi Arabia. Therefore, the aim of this study is to estimate the prevalence, characteristics, applied management, and outcomes of patients with acute appendicitis during COVID-19 in comparison to pre-COVID-19 cases in an academic tertiary center.Study settings and designThis is a retrospective study design aimed to target patients with acute appendicitis presented to King Abdul-Aziz Medical City, Academic Tertiary Center, Jeddah, Saudi Arabia.Identifications of study participantsPatients with acute appendicitis between July 2018 and October 2021 were included. The first case of COVID-19 spotted in Saudi Arabia was in March 2019, and thereafter, it has been declared a worldwide pandemic. While October 2021 was the month when all COVID-19 restrictions were first lifted in Saudi Arabia. Therefore, patients with acute appendicitis were classified into two groups based on whether the pandemic has started or not in Saudi Arabia. So, those with acute appendicitis presenting to the emergency department between July 2018 and February 2020 were classified into a pre-COVID-19 pandemic (group A), while those presenting between March 2020 and October 2021 were under the COVID-19 pandemic (group B). Patients were identified using the International Classification of Diseases (ICD-10) codes. Data collection processThe data were obtained from the electronic medical records of the academic tertiary center using the Best Care 2.0 System . A data collection form was designed and contained the study variables, such as demographic and clinical data. For example, age, gender, period of presentation, and laboratory values were collected. Additionally, the length of symptoms prior to emergency presentation, treatment modalities, and grade of appendicitis were also obtained. Afterward, the collected data were entered into a Microsoft Excel  file by the research team and kept confidential. Statistical analysisContinuous variables were reported using the mean and standard deviation. While categorical variables were reported as frequencies and percentages. Variables were analyzed by the Chi-square test, Fisher’s exact test, and T-test as appropriate. Statistical significance was defined as P<0.05. Statistical analysis was performed using JMP Statistical Software  a subsidiary of SAS Institute, version 15.2.0.Demographics and clinical characteristicsA total of 298 patients were identified and included between the two specified periods. In group A, there were a total of 161 patients , of which 104 (64.6%) were male, and a total of 137 patients  were included in group B, of which 80 (58.4%) were male (P=0.2723). Moreover, the number of patients who presented to the ER between 24 and 48 hours after symptom onset in group A was 111 (68.9%) compared to 89 (65%) in group B  cases in group A, while it was performed in 96 (70.1%) patients in group B, with a statistically significant difference (P=0.0106). In addition, 19 (11.8%) patients had an open appendectomy in group A in contrast to group B with 27 (19.7%) patients, (P=0.0597). Furthermore, the number of patients who re-visited the ER after hospital discharge for a similar presentation was 14 (8.7%) in group A vs 10 (7.3%) in group B (P=0.6769). Only four (2.5%) patients of group A required re-admission vs six (4.4%) patients of group B, (P=0.5213), Table .6% casesLength of symptom and grade of appendicitisFor those who had uncomplicated appendicitis, 135 (83.9%) patients were in group A vs 113 82.5%) in group B, (P=0.7526). Whereas, for perforated appendicitis, eight (5%) cases were found in group A vs 15 (11%) group B cases, (P=0.0797). Also, gangrenous appendicitis in group A was found among 10 (6.2%) patients vs two (1.5%) in group B, with a statistically significant difference (P=0.0420), as shown in Table .5% in grDuring the COVID-19 pandemic, many aspects of clinical practice have significantly changed. Investigating such changes might be beneficial in further understanding the disease outcomes and evaluating the current management practices and plans. This study aimed to investigate the prevalence, characteristics, applied management, and outcomes of patients with acute appendicitis during COVID-19 in comparison to the pre-COVID-19 period.Number of acute appendicitisCompared to the pre-COVID-19 period, our findings demonstrated a reduction in the number of patients presenting with acute appendicitis during the COVID-19 period, from 161 to 137 patients. Such observation was also noted by several other investigators, Scheijmans et al., who reported a reduction in the rate of acute appendicitis among their patients during the pandemic -10. ThisLength of symptomsDue to the limited access to healthcare and reduced number of acute appendicitis presentations, it would be assumed that delayed presentation to the ER would be more common during the COVID-19 period. However, our results showed that there was no statistical difference between the two periods in the length of symptoms prior to presentation, with most patients presenting between 24 and 48 hours of symptom onset in both periods. Bosak Veršić et al. noted that most patients before and during the pandemic presented within the first day . Our finGrades of acute appendicitisOur study results revealed increasing rates of reported perforated appendicitis cases in group B, and this could be a result of the patient's behaviors during the pandemic. Patients’ attitudes toward seeking medical help during COVID-19 have changed, as most of them defer medical consultation if possible. In fact, most of the perforated appendicitis in group B presented to the ER within 48-72 hours and >72 hours of symptoms onset vs those in group A who presented within 24-48 hours. This delay in seeking medical help has been observed by other investigators too, including Somers, El Nakeeb, and Scheijmans et al., and it is most likely to be associated with a more complicated clinical course . Management of acute appendicitisIn regard to management, this study has found a statistically significant difference between both periods. A higher chance of undergoing laparoscopic appendectomy was noted among group A patients than those in group B. Similarly, a meta-analysis reported that the rate of open appendectomies during the pandemic was higher in comparison to laparoscopic procedures . Since lER re-visit and re-admissionThis study found no significant differences in the number of patients re-visiting the ER with similar symptoms after discharge or in the rates of re-admission between the two periods. Re-admission rate was also noted to be similar by other studies compared to the two periods ,17. El NLimitations and recommendationsIt is a single-center study that limits the generalizability of the results in regard to the influence of the COVID-19 pandemic on acute appendicitis. Therefore, a larger scale study would be beneficial in which it may involve all centers of Saudi Arabia. Also, addressing patients' and the public's concerns are vitally important as well as educating them regarding the risk of not seeking timely appropriate care for such a health condition due to the raised concerns from pandemics.Finally, during the COVID-19 period, there was a reduction in the number of patients presenting with acute appendicitis, with a lower chance of undergoing laparoscopic appendectomy. There was an increase in perforated appendicitis and a decrease in gangrenous appendicitis.Therefore, having current data regarding the trends of acute appendicitis is highly crucial, as it gives insight into the effect of such a pandemic and provides lessons to be learned in preparation for future pandemics. This study mainly presents a Saudi perspective on changing trends in acute appendicitis that would help Middle Eastern and international researchers in exploring the effect of the COVID-19 pandemic on such important emergency surgical cases.